Impact of different sterilisation techniques on sorption and NER formation of test chemicals in soil.

Standard OECD tests are used to generate data on biodegradation (OECD 307) and sorption (OECD 106) of test chemicals in soil. In such tests, data on abiotic degradation using sterile samples are utilised to investigate any losses due to abiotic processes. The data from sterile samples are also used to interpret results and findings of non-sterile samples, especially in the context of sorption and non-extractable residue (NER) formation. However, to ensure the comparability of the data obtained from sterile and non-sterile experiments, effects of sterilisation on the soil matrix should be minimal. The objective of this study was to investigate the efficiencies of different sterilisation techniques and the impact of the sterilisation on sorption and NER formation in soil. In this study, experiments in accordance with OECD 307 and OECD 106 guidelines were performed with two soils covering wide range of soil characteristics and treated with the three sterilisation techniques autoclaving, gamma(γ)-radiation and adding 1% (w/w) sodium azide. As a test item, 14C-labelled phenanthrene and bromoxynil was used for OECD 307 test, whereas non-labelled phenanthrene and atrazine was used for OECD 106. The sterilisation efficiencies were investigated using traditional viable plate count and molecular approaches (RNA extraction method). The results suggest that none of the tested techniques resulted in completely sterilised soil with autoclaving being the most efficient technique. Adding sodium azide led to most inefficient sterilisation and a significant increase (0.56 units) in soil pH. OECD 307 results showed differences in NER formation of the test chemicals, especially for soil poisoning and γ-radiation, which could be due to inefficient sterilisation and/or change in soil physico-chemical properties. OECD 106 results suggest that none of the sterilisation techniques considerably affected sorption behaviour of the test chemicals. Based on our results, we recommend autoclaving as most suitable sterilisation technique.

Improved closed test setup for biodegradation testing of slightly volatile substances in water-sediment systems (OECD 308).

Standardized biodegradation testing methods, like the OECD 308 Aerobic and Anaerobic Transformation in Aquatic Sediment Systems, generate data on biodegradation required during environmental risk and hazard assessment of chemicals under different European and international regulations. However, difficulties arise when applying the OECD 308 guideline for testing hydrophobic volatile chemicals. Especially the use of a co-solvent (like acetone) as a measure to facilitate the application of the test chemical in combination with a closed setup to reduce losses due to volatilization tend to deplete/restrict the amount of oxygen in the test system. The result is a low oxygen or even anoxic water column in the water-sediment system. Thus, the degradation half-lives of the chemical generated from such tests are not directly comparable to the regulatory half-life values for Persistence assessment of the test chemical. The aim of this work was to further develop the closed setup to improve and maintain aerobic conditions in the water phase of the water-sediment systems for testing slightly volatile hydrophobic test chemicals. This improvement was attained by optimizing the test system geometry and agitation technique to maintain aerobic conditions in the water phase in a closed test setup, investigating appropriate co-solvent application strategy, and trialing the resulting test setup. This study shows that when using a closed test setup for OECD 308 tests, agitation of the water phase overlaying the sediment and the test item application using low co-solvent volume is critical for maintaining an aerobic water layer.

Biodegradation testing of volatile hydrophobic chemicals in water-sediment systems - Experimental developments and challenges.

Degradation data are crucial for the persistence assessment of chemicals and they are generated using standard OECD guidelines. The OECD 308 describes a simulation biodegradation test of chemicals in water-sediment systems. This guideline is not applicable for testing highly volatile chemicals and recommends a closed biometer test setup for testing slightly volatile chemicals. However, proper details on system geometries, construction and monitoring of aerobic conditions are not provided. The choice of system geometry and sediment:water ratio influences the partitioning of test chemicals between different compartments (water, sediment and headspace) and can therefore affect their degradation. The guideline recommends the addition of test chemical via aqueous solutions, which however is not possible for hydrophobic volatile chemicals due to their volatilization losses and low solubility. Thus, the use of a co-solvent is necessary for the application of such chemicals but its effects in a closed setup has not been studied. We recently developed an improved closed test setup for testing volatile chemicals in soil. The objective was to adapt this improved test setup to conduct OECD 308 tests using 14C labelled chemicals with different volatilities. Using the adapted test setup it was possible to obtain a complete mass balance even for n-decane and tetralin having the highest Henry's constants of the tested chemicals. However, the use of co-solvent affected the oxygen levels, which in turn affected microbial activity and likely also the degradation of test chemicals. Therefore, the adapted test setup needs further developments for the testing of volatile hydrophobic chemicals.

Biodegradation of Volatile Chemicals in Soil: Separating Volatilization and Degradation in an Improved Test Setup (OECD 307).

During environmental risk assessments of chemicals, higher-tier biodegradation tests in soil, sediment, and surface-water systems are required using OECD standards 307, 308, and 309 guidelines, respectively. These guidelines are not suitable for testing highly volatile chemicals, and a biometer closed-incubation setup is recommended for testing slightly volatile chemicals. In this setup, the degradation kinetics of highly volatile chemicals can largely be influenced by volatilization. Additionally, guidelines lack sufficient information on test-system geometry and guidance on how to measure and maintain aerobic conditions during the test. Our objectives were (1) to design a closed test setup for biodegradation tests in soil in which the maintaining and measuring of aerobic conditions was possible without the loss of volatile test chemicals and (2) to suggest data-treatment measures for evaluating the degradation kinetics of volatile test chemicals. With the new setup, full-scale OECD 307 tests were performed using the volatile 14C-labeled chemicals decane and tetralin. For both test chemicals, reproducible complete mass balances were observed, and the new setup ensured that the volatilization losses were kept below the mineralized fraction. Based on the obtained data, an extended model was developed that enabled consideration of the volatilization in the modeling of degradation kinetics.

Simulation Studies to Explore Biodegradation in Water-Sediment Systems: From OECD 308 to OECD 309.

Studies according to OECD 308 and OECD 309 are performed to simulate the biodegradation of chemicals in water-sediment systems in support of persistence assessment and exposure modeling. However, several shortcomings of OECD 308 have been identified that hamper data evaluation and interpretation, and its relation to OECD 309 is still unclear. The present study systematically compares OECD 308 and OECD 309 and two variants thereof to derive recommendations on how to experimentally address any shortcomings and improve data for persistence and risk assessment. To this end, four (14)C-labeled compounds with different biodegradation and sorption behavior were tested across standard OECD 308 and 309 test systems and two modified versions thereof. The well-degradable compounds showed slow equilibration and the least mineralization in OECD 308, whereas the modified systems provided the highest degree of mineralization. Different lines of evidence suggest that this was due to increased oxygenation of the sediment in the modified systems. Particularly for rapidly degrading compounds, non-extractable residue formation was in line with degradation and did not follow the sediment-water ratio. For the two more slowly degrading compounds, sorption in OECD 309 (standard and modified) increased with time beyond levels proposed by equilibrium partitioning, which could be attributed to the grinding of the sediment through the stirring of the sediment suspension. Overall, the large differences in degradation observed across the four test systems suggest that refined specifications in test guidelines are required to reduce variability in test outcomes. At the same time, the amount of sediment and its degree of oxygenation emerged as drivers across all test systems. This suggests that a unified description of the systems was possible and would pave the way toward a more consistent consideration of degradation in the water-sediment systems across different exposure situations and regulatory frameworks.

Bridging across OECD 308 and 309 Data in Search of a Robust Biotransformation Indicator.

The OECD guidelines 308 and 309 define simulation tests aimed at assessing biotransformation of chemicals in water-sediment systems. They should serve the estimation of persistence indicators for hazard assessment and half-lives for exposure modeling. Although dissipation half-lives of the parent compound are directly extractable from OECD 308 data, they are system-specific and mix up phase transfer with biotransformation. In contrast, aerobic biotransformation half-lives should be easier to extract from OECD 309 experiments with suspended sediments. Therefore, there is scope for OECD 309 tests with suspended sediment to serve as a proxy for degradation in the aerobic phase of the more complicated OECD 308 test, but that correspondence has remained untested so far. Our aim was to find a way to extract biotransformation rate constants that are universally valid across variants of water-sediment systems and, hence, provide a more general description of the compound's behavior in the environment. We developed a unified model that was able to simulate four experimental types (two variants of OECD 308 and two variants of OECD 309) for three compounds by using a biomass-corrected, generalized aerobic biotransformation parameter (k'bio). We used Bayesian calibration and uncertainty assessment to calibrate the models for individual experimental types separately and for combinations of experimental types. The results suggested that k'bio was a generally valid parameter for quantifying biotransformation across systems. However, its uncertainty remained significant when calibrated on individual systems alone. Using at least two different experimental types for the calibration of k'bio increased its robustness by clearly separating degradation from the phase-transfer processes taking place in the individual systems. Overall, k'bio has the potential to serve as a system-independent descriptor of aerobic biotransformation at the water-sediment interface that is equally and consistently applicable for both persistence and exposure assessment purposes.